The Phospholipase D (PLD) catalyzed conversion of phosphatidylcholine to choline and phosphatidic acid (PA) is a key regulatory step in a wide array of signaling pathways. Two PLD isoforms have been identified based upon their specificity for activators. PLD1 is activated by RhoA, protein kinase C (PKC), and Arf, while PLD2 has a substantial basal level of activity but may be regulated by Arf and PKC. Preliminary research in our laboratory has identified a PLD activity in Chinese hamster lung fibroblasts (CCL39) that is stimulated by the alphal-adrenergic agonist phenylephrine (PE). We have previously shown that PE stimulation of CCL39 cells leads to the activation of the Na-H exchanger isoform 1(NHE1), RhoA and extracellular signal regulated kinase (Erk). Furthermore, PE stimulation induces the formation of stress fibers. Subsequent experiments suggested that the PE induced Erk activation can be blocked by dominant negative RhoA. Additionally, stress fiber formation is blocked when the activity of NHE1, RhoA or Erk is inhibited. These preliminary results have lead us to the hypothesis that the regulation of NHE1 by alphaladrenergic activation is mediated by a RhoA-dependent PLD in CCL39 fibroblasts. Specifically, we propose that PE activates RhoA in a PKC dependent manner. RhoA then activates PLD1, which in turn increased the levels of PA. The increase in PA then leads to the activation of the Ras-Erk pathway by stimulating Raf. To delineate the role of PLD is the regulation of NHE1 activity and a potential role in stress fiber formation and cell migration, this project aims to investigate: 1) Which isoform of PLD is present in CCL39 cells that can be activated by PE; 2) Determine the importance of PLD in Erk and NHE1 regulation and subsequently how regulation of Erk and NHE relate to the control of stress fiber formation and cell migration; and 3) Characterize the mechanism of RhoA and PLD activation of Erk and NHE1 in CCL39 Cells. In short, this proposal focuses on an important potential role for PLD and will help increase the understanding of the mechanism of PLD function. [unreadable] [unreadable] Finally, this research will be done at a predominantly undergraduate institution, Minnesota State University Moorhead. All of the preliminary data was collected by undergraduates and the funding of this project would involve 15 to 20 undergraduates in meaningful research over the three-year term of the grant. [unreadable] [unreadable]